The present invention relates to a novel topoisomerase IV, the nucleotide sequences encoding this enzyme, their corresponding vectors and the use of this enzyme for screening biologically active products.
Topoisomerases are enzymes capable of modifying the topological configuration of DNA rings, of making knots therein or of interlacing separated rings. They are thus involved in the replication, transcription and recombination of the entire genetic information (Wang et al., 1990). The mechanism of all these topological conversions is the same: the ring is opened so that a segment of DNA passes through the gap before the ends are rejoined. Two types of topoisomerase are involved in these conversions: type I topoisomerases which cut a single DNA strand and type II topoisomerases which cut both strands simultaneously.
Up until now, two type II bacterial topoisomerases have been identified and studied more particularly: gyrase from Escherichia coli (Gellert et al., 1976), and more recently, DNA topoisomerase IV from E. coli (Kato et al., 1990).
Gyrase is a xcex12xcex22 tetramer whose xcex1 or GyrA and xcex2 or GyrB subunits are encoded by the gyrA and gyrB genes respectively. Bacterial gyrases are the only known topoisomerases capable of supercoiling relaxed DNA rings in the presence of ATP.
As regards more particularly DNA topoisomerase IV from E. coli, it relaxes supercoiled plasmid DNA, unknots T4 phage DNA and unwinds (or decatenates) kinetoplast DNA (Kato et al., 1992; Peng et al., 1993). The sequence of its corresponding genes, parC and parE from E. coli, has made it possible to demonstrate regions of high similarity between the subunits of gyrase and those of this topoisomerase IV, ParC with GyrA (35.6% over the entire sequence) and ParE with GyrB (40.1% over the entire sequence) respectively (Kato et al., 1990).
E. coli gyrase has also been identified as being a primary target of fluoroquinolones (Hooper et al., 1993). It has thus been demonstrated that E. coli strains mutated at the level of the Ser83 residue in the GyrA subunit have a high resistance to fluoroquinolones (Maxwell, 1992). Fluoroquinolones bind less to DNA-mutated gyrase complexes than to DNA-wild-type gyrase complexes. Indeed, other point mutations, mapped in the region between residues 67 and 106 of GyrA, lead to strains resistant to fluoroquinolones. This region is called QRDR (Yoshida et al., 1990; Cullen et al., 1989). Similar results have been published with strains of Staphylococcus aureus resistant to fluoroquinolones (Goswitz et al., 1992; Sreedharan et al., 1990). Gyrase is therefore nowadays recognized as being the primary target of quinolones. However, a clinical strain of Staphylococcus aureus, not containing any mutation in the QRDR region of GyrA, has also been described as resistant to fluoroquinolones (Sreedharan et al., 1991).
Nowadays, this phenomenon of resistance developed by Staphylococcus aureus bacteria towards antibiotics and more particularly towards fluoroquinolones is being increasingly encountered at the therapeutic level. It would be particularly important to be able to lift this resistance and this involves a characterization of all the parameters which are associated with it.
The main objective of the present invention is precisely the identification, sequencing and characterization of nucleic sequences encoding subunits of a novel topoisomerase, topoisomerase IV of Staphylococcus aureus, composed of two subunits, GrlA and GrlB.
Unexpectedly, the applicant has found that the primary target of the fluoroquinolones in S. aureus is a topoisomerase IV and not gyrase. It has thus demonstrated that clinical strains of S. aureus, in which the QRDR region of the GyrA subunit of gyrase is identical to the wild-type sequence, develop nevertheless a resistance to fluoroquinolones because of a mutation which they possess in the region of the GrlA subunit of topoisomerase IV, homologous to the QRDR region.
The first subject of the present invention is a nucleotide sequence encoding at least one subunit of topoisomerase IV of Staphylococcus aureus. 
The present invention describes in particular the isolation and the characterization of the grlA and grlB genes. These genes have been cloned, sequenced and expressed in E. coli, and their enzymatic activity has been characterized. They were isolated from a Staphylococcus aureus genomic DNA library. From the grlAB nucleic sequence (SEQ ID No. 1 and SEQ ID No. 2), two open frames, corresponding to the grlB and grlA genes respectively, have been identified. The grlA and grlB genes have been sequenced in SEQ ID No. 4 and SEQ ID No. 6 respectively.
Preferably, the subject of the present invention is a nucleotide sequence chosen from:
(a) all or part of the grlA (SEQ ID No. 4) or grlB (SEQ ID No. 6) genes,
(b) the sequences hybridizing with all or part of the (a) genes and encoding a subunit of a topoisomerase IV, and
(c) the sequences derived from the (a) and (b) sequences because of the degeneracy of the genetic code.
It is clear that from the genes identified in the present application, it is possible, by hybridization, to directly clone other genes encoding a subunit of topoisomerase IV of bacteria close to S. aureus such as for example Streptococci and Enterococci. It is thus possible to clone this type of gene using, as probe, the genes grlA, grlB or fragments thereof. Likewise, the cloning of these genes may be carried out using degenerate oligonucleotides derived from sequences of the grlA or grlB genes or fragments thereof.
For the purposes of the present invention, derivative is understood to mean any sequence obtained by one or more modifications and encoding a product conserving at least one of the biological properties of the original protein. Modification should be understood to mean any mutation, substitution, deletion, addition or modification of a genetic and/or chemical nature. These modifications may be performed by techniques known to persons skilled in the art.
Among the preferred derivatives, there may be mentioned more particularly natural variants, molecules in which one or more residues have been substituted, derivatives obtained by deletion(s) of regions not or little involved in the interaction between the binding sites considered or expressing an undesirable activity, and derivatives having, compared with the native sequence, one or more additional residues.
Still more preferably, the subject of the invention is the nucleotide sequences represented by the grlA (SEQ ID No. 4) and grlB (SEQ ID No. 6) genes.
It also relates to any grlA gene having a mutation leading to a resistance to molecules of the quinolone and more particularly of the fluoroquinolone family. As a representative of these mutated genes, there may be mentioned more particularly the grlA gene having a base change from C to A at position 2270 of SEQ ID No. 4. The resulting gene is termed grlA(C-2270A). This mutation leads to substitution of the Ser-80 residue with Tyr in the GrlA protein. The resulting protein will be designated by GrlA(Ser-80 Tyr).
Another subject of the present invention relates to a recombinant DNA comprising at least one nucleotide sequence encoding a subunit of topoisomerase IV of Staphylococcus aureus. More particularly, it is a recombinant DNA comprising at least one nucleotide sequence as defined above in (a), (b) and (c) and more particularly the gene grlA (SEQ ID No. 4) grlA(C-2270A) and/or the gene grlB (SEQ ID No. 6).
According to a preferred mode of the invention, the nucleotide sequences defined above form part of an expression vector which may be autonomously replicating or integrative.
Another subject of the invention relates to the polypeptides resulting from the expression of the nucleotide sequences as defined above. More particularly, the present invention relates to the polypeptides comprising all or part of the polypeptides GrlA (SEQ ID No. 2) or GrlB (SEQ ID No. 3) or of their derivatives. For the purposes of the present invention, the term derivative designates any molecule obtained by modification of the genetic and/or chemical nature of the peptide sequence. Modification of the genetic and/or chemical nature may be understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for different purposes, such as especially that of increasing the affinity of the peptide for its substrate(s), that of enhancing its production levels, that of increasing its resistance to proteases, that of increasing and/or of modifying its activity, or that of conferring new biological properties on it. Among the derivatives resulting from an addition, there may be mentioned, for example, the chimeric polypeptides containing an additional heterologous part attached to one end. The term derivative also comprises the polypeptides homologous to the polypeptides described in the present invention, derived from other cellular sources.
Preferably, they are the polypeptides GrlA (SEQ ID No. 3), GrlB (SEQ ID No. 5) and GrlA(Ser-80Tyr).
The subject of the invention is also any recombinant cell containing a nucleotide sequence, a recombinant DNA and/or a vector as defined above. The recombinant cells according to the invention may be both eukaryotic and prokaryotic cells. Among the suitable eukaryotic cells, there may be mentioned animal cells, yeasts, or fungi. In particular, as regards yeasts, there may be mentioned yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula. As regards animal cells, there may be mentioned COS, CHO and C127 cells, Xenopus eggs, and the like. Among the fungi, there may be mentioned more particularly Micromonospora, Aspergillus ssp. or Trichoderma ssp. Preferably, they are prokaryotic cells. In this case, the following bacteria may be more particularly used: Actinomycetes, Bacillus, and more preferably E. coli and Staphylococcus. The recombinant cells of the invention may be obtained by any method allowing the introduction of a foreign nucleotide sequence into a cell. This may be especially transformation, electroporation, conjugation, fusion of protoplasts, or any other technique known to persons skilled in the art.
The subject of the present invention is also a process for the preparation of polypeptides as claimed from the culture of one of these recombinant cells. The polypeptide(s) thus obtained are recovered according to conventional methods after the culture.
The invention also relates to an isolated topoisomerase IV capable of being obtained from the expression of all or part of the grlA gene (SEQ ID No. 4) and of all or part of the grlB gene (SEQ ID No. 6) or of their respective derivatives.
Derivative is understood to designate the sequences hybridizing with all or part of the grlA or grlB gene and encoding a subunit of a topoisomerase IV as well as all the sequences derived from a degeneracy of the genetic code of these hybrid sequences or of the sequences corresponding to all or part of the grlA or grlB gene.
More preferably, it is an isolated topoisomerase IV derived from the expression of all or part of the grlA gene (SEQ ID No. 4) or of all or part of the grlB gene (SEQ ID No. 6).
The present invention relates more particularly to any topoisomerase IV behaving as a primary target towards fluoroquinolones.
According to a specific mode of the invention, it is topoisomerase IV of Staphylococcus aureus. 
The claimed topoisomerase IV according to the invention is most particularly useful for targeting biologically active products such as for example potential antibiotics and especially molecules of the fluoroquinolone family. Advantageously, it may also be used to assay and/or identify products inhibiting the ATP-dependent DNA relaxing reaction and/or the products inhibiting the reaction of decatenation of catenanes of DNA.
The applicant has thus developed an assay for enzymatic activity which is specific for topoisomerase IV of S. aureus and has shown that this activity is inhibited by antibiotic molecules such as fluoroquinolones.
The present invention provides a new target for searching for new antibiotics, as well as a screen specific for this target; this screen is described in Example 7. This screen makes it possible to demonstrate the products which inhibit DNA topoisomerase IV of S. aureus. The following may be used in this test: pure or mixed synthetic products, natural plant extracts, bacterial cultures, fungi, yeasts or algae, pure or in the form of a mixture. The test described in the present invention makes it possible to detect both products which stabilize the cleavable complex, a reaction intermediate of the reaction catalysed by the enzyme, and also inhibitors acting through other mechanisms.